EarlyTox Cell Integrity Kit The EarlyTox Cell Integrity Kit from Molecular Devices is an optimized set of reagents that simplifies the measurement of live and dead cells in a single well. The assay uses two nuclear dyes that allow users to detect changes in outer cell membrane permeability resulting from cell damage or cell death via necrosis, apoptosis, or other mechanisms. Cell viability is assessed by counting the number or percent of live and damaged cells. The assay can be used for studies of the effects of different treatments on cell viability, evaluation of toxicity effects of pharmaceutical compounds or other chemicals, studies of necrosis and apoptosis, and many other applications. The EarlyTox Cell Integrity Kit is designed to work with many cell types, both adherent and non-adherent. The simple workflow and reagent performance of the protocol make it amenable to high-throughput screening. Table 1-1: Available Kits Assay Kit Explorer Kit Bulk Kit EarlyTox Cell Integrity Kit (200 Tests, two (2) 96-well or 384-well microplates) R8213 EarlyTox Cell Integrity Kit (1000 Tests, ten (10) 96-well or 384-well microplates) R8214 Contents Chapter 1: About the EarlyTox Cell Integrity Kit 3 Assay Principle 3 Chapter 2: Materials and Equipment 4 Kit Components 4 Materials Required but Not Provided 4 Storage and Handling 4 Chapter 3: Assay Protocols 5 Standard Protocol 5 Homogeneous Assay Protocol 6 Setup for the SpectraMax MiniMax 300 Imaging Cytometer 7 Setup for the ImageXpress Micro XLS System 9 Chapter 5: Data Analysis Examples 11 Chapter 6: Obtaining Support 13 R3614 A March 2014 www.moleculardevices.com
This document is provided to customers who have purchased Molecular Devices equipment, software, reagents, and consumables to use in the operation of such Molecular Devices equipment, software, reagents, and consumables. This document is copyright protected and any reproduction of this document, in whole or any part, is strictly prohibited, except as Molecular Devices may authorize in writing. Software that may be described in this document is furnished under a non-transferrable license. It is against the law to copy, modify, or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the license agreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. Portions of this document may make reference to other manufacturers and/or their products, which may contain parts whose names are registered as trademarks and/or function as trademarks of their respective owners. Any such usage is intended only to designate those manufacturers products as supplied by Molecular Devices for incorporation into its equipment and does not imply any right and/or license to use or permit others to use such manufacturers and/or their product names as trademarks. Each product is shipped with documentation stating specifications and other technical information. Molecular Devices products are warranted to meet the stated specifications. Molecular Devices makes no other warranties or representations express or implied, including but not limited to, the fitness of this product for any particular purpose and assumes no responsibility or contingent liability, including indirect or consequential damages, for any use to which the purchaser may put the equipment described herein, or for any adverse circumstances arising therefrom. The sole obligation of Molecular Devices and the customer's sole remedy are limited to repair or replacement of the product in the event that the product fails to do as warranted. For research use only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of Molecular Devices, LLC or their respective owners. These trademarks may not be used in any type of promotion or advertising without the prior written permission of Molecular Devices, LLC. Patents: http://www.moleculardevices.com/productpatents Product manufactured by Molecular Devices, LLC. 1311 Orleans Drive, Sunnyvale, California, United States of America 94089. Molecular Devices, LLC is ISO 9001 registered. 2014 Molecular Devices, LLC. All rights reserved. 2 R3614 A
EarlyTox Cell Integrity Kit Chapter 1: About the EarlyTox Cell Integrity Kit The EarlyTox Cell Integrity Kit provides a fast, simple, and reliable fluorescence-based assay for detecting changes in cell viability based on permeability of outer cell membranes (cell integrity). The kit is based on two nuclear dyes: Live Red Dye is cell permeant and marks both live and dead cells (Excitation: 622 nm/emission: 645 nm). Dead Green Dye is cell impermeant and stains only cells with damaged outer membranes (Excitation: 503 nm/emission: 526 nm). Note: Use the appropriate wavelength setting for your instrument. See Setup for the SpectraMax MiniMax 300 Imaging Cytometer on page 7 or Setup for the SpectraMax MiniMax 300 Imaging Cytometer on page 7. The kit also contains a proprietary EarlyTox Masking Reagent to increase assay performance in a homogeneous format. Each kit enables a simple mix-and-read procedure whereby cells are incubated with reagents for 30 minutes and then transferred directly to an imaging instrument for evaluation. Time-consuming wash steps are eliminated. Assay Principle Live Red Dye Dead Green Dye Live Cell Cell-permeant Live Red Dye crosses the intact membranes of live cells. It then binds to DNA and becomes highly fluorescent. Cell-impermeant Dead Green Dye is excluded from live cells. Dead Cell The membrane integrity of dead and dying cells is compromised, allowing Dead Green Dye to enter. Both dyes are now bound to DNA, so dead cells exhibit both red and green fluorescence. Figure 1-1: EarlyTox Cell Integrity Kit assay principle R3614 A 3
Chapter 2: Materials and Equipment Kit Components Table 2-1: Components of the EarlyTox Cell Integrity Kits Item Explorer Kit (R8213) Bulk Kit (R8214) EarlyTox Live Red Dye 1 vial 1 vial EarlyTox Dead Green Dye 1 vial 1 vial EarlyTox Masking Reagent 1 bottle 3 bottles The entire EarlyTox Cell Integrity Explorer Kit (R8213) is sufficient for two (2) 96-well or 384-well microplates. The entire EarlyTox Cell Integrity Bulk Kit (R8214) is sufficient for ten (10) 96-well or 384-well microplates. Materials Required but Not Provided Table 2-2: Reagents and Supplies Item DPBS Buffer (1x Dulbecco s Phosphate Buffered Saline) ph 7.4 Suggested Vendor Corning Cellgro or equivalent Storage and Handling On receipt of the EarlyTox Cell Integrity Kit, store the contents at 20 C. Under these conditions the reagents are stable for six months in the original packaging. WARNING! Reagents can contain chemicals that are harmful. Exercise the applicable care when handling reagents as described in the related safety data sheet (SDS or MSDS). The safety data sheet is available in the Knowledge Base on the Molecular Devices support web site: www.moleculardevices.com/support.html 4 R3614 A
Chapter 3: Assay Protocols Standard Protocol 1. Plate the cells at the desired plating densities (generally overnight) and treat the cells with compounds according to the specific experiment (generally for 24 to 48 hours). Note: For imaging applications, use black-well, clear-bottom microplates. 2. Prepare 1:2000 dilutions of the dyes (add 10 µl of each reagent to 20 ml of DPBS) immediately before cell staining. 3. Gently remove the cell media from the microplates by pipetting or aspirating (do not wash the cells). 4. Add the staining solution directly into the wells. Add 100 µl per well for 96-well microplates. Add 25 µl per well for 384-well microplates. 5. Return the microplates to the incubator and incubate for 15 to 30 minutes at 37 C and 5% CO 2. 6. Proceed with image acquisition using the appropriate wavelengths for the Live Red Dye and Dead Green Dye. See Setup for the SpectraMax MiniMax 300 Imaging Cytometer on page 7 or Setup for the ImageXpress Micro XLS System on page 9. Live Red Dye can be detected using the 713 channel (Ex: 625 nm/em: 713 nm). Dead Green Dye can be detected by using the 541 channel (Ex: 456 nm/em: 541 nm). Typical exposure times are 10 to 20 ms. Note: Adjust the exposure times for the 713 and 541 channels to achieve the correct color balance between channels. Generally, 541 exposure time is 2x greater than 713 exposure time. R3614 A 5
Homogeneous Assay Protocol Note: In this protocol the culture medium is not removed from the wells. The optional Masking Reagent can be used to reduce the background fluorescence typically created by the medium. 1. Grow cells in 96-well or 384-well microplates with the appropriate treatments. The final volume of media should be approximately 100 µl for 96-well microplates and 25 µl for 384-well microplates. 2. Prepare 2x (1:1000) solution of staining reagents in DPBS (for example, 10 µl of each reagent in 10 ml of DPBS) immediately before cell staining. 3. Add a volume of 2x staining solution (equal to the volume already in the well) directly to the wells. Add 100 µl for 96-well microplates. Add 25 µl for 384-well microplates. 4. Return the microplates to the incubator and incubate for 30 minutes at 37 C and 5% CO 2. 5. Optional: Prepare a 5x solution of Masking Reagent (add 10 ml of DPBS to the bottle and vortex for ~1 minute). Add the 5x solution of Masking Reagent directly into the media (25 µl per 96-well microplate, 6.3 µl per 384-well microplate). Image the plate immediately. Note: Do not add Masking Reagent simultaneously with staining solution. 6. Proceed with image acquisition using the appropriate wavelengths for the Live Red Dye and Dead Green Dye. See Setup for the SpectraMax MiniMax 300 Imaging Cytometer on page 7 or Setup for the ImageXpress Micro XLS System on page 9. Typical exposure time 10 to 20 ms. If Masking Reagent is used, it is necessary to increase imaging exposure times (100 to 200 ms). Note: For optimum results it is important to use the recommended reagent volumes and concentrations. However, standard procedures vary across laboratories, and Molecular Devices recognizes that a variety of cell-handling conditions might be adopted at the discretion of the user. Assay development might be required for optimization. Table 3-1: Suggested Plating Volumes and Seeding Densities Cell Type 96-well microplate (100 µl growth medium with compounds) 384-well microplate (25 µl growth medium with compounds) Adherent cells 4000 to 20000 (cells per well) 1000 to 5000 (cells per well) Non-adherent cells 20000 to 100000 (cells per well) 5000 to 20000 (cells per well) 6 R3614 A
Setup for the SpectraMax MiniMax 300 Imaging Cytometer Acquire images from the prepared microplate using the SpectraMax MiniMax 300 Imaging Cytometer and the SoftMax Pro Microplate Data Acquisition and Analysis Software from Molecular Devices with the following suggested settings. Wavelength: Excitation: 456 nm / Emission: 541 nm for Dead Green Dye Excitation: 625 nm / Emission: 713 nm for Live Red Dye Image Acquisition Settings: Select control and treated wells for the Max Image and Min Image wells For Live Red Dye (713 nm), use a typical exposure time of 10 to 20 ms For Dead Green Dye (541 nm), use a typical exposure time of 20 ms Image Analysis Settings: Use Discrete Object Analysis Wavelength for Finding Objects: 713 nm Find Objects Finding Method: Draw on Images, or Set Size and Intensity Note: If you are using Masking Reagent, select Set Size and Intensity. Draw on Images settings: Nuclei preset drawing analysis Set Size and Intensity settings: Min. Width: 5 µm Max. Width: 30 µm Intensity Above Background: 100 200 (adjust as needed for best results) Click Apply Classification Settings: Create New Classification Type A: Rename to Live Select the objects that are red but not green. Type B: Rename to Dead Select the objects that are both green and red. Click Apply Measurements Default (all parameters selected) Click OK to save the acquisition and analysis settings. R3614 A 7
Click Read to acquire the images with the defined analysis settings. In the Plate section, define a template for the tested compounds and concentrations. Create appropriate tables and graphs using the desired output parameters. Use SoftMax Pro Software or another program for additional analysis. Figure 3-1: Example of Results of Classification for EarlyTox Cell Integrity Assay Data Analysis Using the SoftMax Pro Software 8 R3614 A
Setup for the ImageXpress Micro XLS System Acquire images from the prepared microplate using the ImageXpress Micro XLS Widefield High Content Analysis System and the MetaXpress High Content Image Acquisition and Analysis Software from Molecular Devices with the following suggested configurations and settings. 20x, 10x, or 4x objectives Cy5 filter cube (Ex: 628 nm, Em: 692 nm) for Live Red Dye with a typical exposure time of 10 ms FITC filter cube (Ex: 485 nm, Em: 530 nm) for Dead Green Dye staining with a typical exposure time of 20 ms For 384-well microplates with the 10x or 20x objectives, take one (1) or more images per well For 96-well microplates with the 10x or 20x objectives, take two (2) to four (4) images per well For the 4x objective, take one (1) image per well For image analysis use the MetaXpress Software with the Live-Dead image processing module. The images are first segmented into individual cells using the Live Red nuclear stain and designated Live or Dead based on the presence of the Dead Green stain. Typical settings for image analysis include the following: Wavelength 1 Parameters (for Live Red Dye): Wavelength 1 Source: Cy5 Approximate min width: 7 µm Approximate max width: 30 µm Intensity above local background: ~500 graylevels Wavelength 2: Parameters (for Dead Green Dye): Wavelength 1 Source: FITC Approximate min width: 5 µm Approximate max width: 30 µm Intensity above local background: ~500 graylevels Note: Nuclear sizes and brightness of staining can vary depending on specific cells and between experiments. The suggested settings should be adjusted for the specific experiment. R3614 A 9
Figure 4-1: Suggested settings for EarlyTox Cell Integrity Assay Data Analysis using the MetaXpress Software and the Live-Dead application module In the Configure Summary Log dialog, select the following parameters for toxicity evaluation: Total Cells (number of all cells, nuclear count) Live Cells Dead Cells % Live Cells % Dead Cells 10 R3614 A
Chapter 5: Data Analysis Examples The figure above shows examples of 4-parametric curve fits for concentration dependence of % of intact (left) or damaged (right) cells. Concentration responses are presented for staurosporine (red), IC 50 0.2 µm; and mitomycin C (green), IC 50 3.4 µm. R3614 A 11
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Chapter 6: Obtaining Support Molecular Devices is a leading worldwide manufacturer and distributor of analytical instrumentation, software and reagents. We are committed to the quality of our products and to fully supporting our customers with the highest possible level of technical service. Our support web site, www.moleculardevices.com/support.html, has a link to the Knowledge Base with technical notes, software upgrades, safety data sheets, and other resources. If you do not find the answers you are seeking, follow the links to the Technical Support Service Request Form to send an email message to a pool of technical support representatives. You can contact your local representative or contact Molecular Devices Technical Support by telephone at 800-635-5577 (U.S. only) or +1 408-747-1700. In Europe call +44 (0) 118 944 8000. Please have the product name, part number, and lot number available when you call. R3614 A 13
Contact Us Phone: Web: +1-800-635-5577 www.moleculardevices.com info@moldev.com Email: Check our website for a current lis ng of worldwide distributors. Regional Offices USA and Canada La n America China (Beijing) China (Shanghai) China (Hong Kong) +1-800-635-5577 +55 11 3616 6607 +86 10 6410 8669 +86 21 3372 1088 +852 3971 3520 Japan (Tokyo) Japan (Osaka) South Korea United Kingdom Germany/Austria +81 3 6362 5260 +81 6 7174 8831 +82 2 3471 9531 +44 (0) 118 944 8000 +49 (0) 89 9605 880 FOR RESEARCH ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The trademarks men oned herein are the property of Molecular Devices, LLC or their respec ve owners. Patents: h p://www.moleculardevices.com/productpatents/ 2014 Molecular Devices, LLC. All rights reserved. www.moleculardevices.c om